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Identification of structural variants (SVs) breakpoints is important in studying mutations, mutagenic causes, and functional impacts. Next-generation sequencing and whole-genome optical mapping are extensively used in SV discovery and characterization. However, multiple platforms and computational approaches are needed for comprehensive analysis, making it resource-intensive and expensive. Here, we propose a strategy combining optical mapping and cas9-assisted targeted nanopore sequencing to analyze SVs. Optical mapping can economically and quickly detect SVs across a whole genome but does not provide sequence-level information or precisely resolve breakpoints. Furthermore, since only a subset of all SVs is known to affect biology, we attempted to type a subset of all SVs using targeted nanopore sequencing. Using our approach, we resolved the breakpoints of five deletions, five insertions, and an inversion, in a single experiment., (© 2022. The Author(s).)

The most prevalent microdeletion in humans occurs at 22q11.2, a region rich in chromosome-specific low copy repeats (LCR22s). The structure of this region has defied elucidation due to its size, regional complexity, and haplotype diversity, and is not well represented in the human genome reference. Most individuals with 22q11.2 deletion syndrome (22q11.2DS) carry a de novo hemizygous deletion of ~ 3 Mbp occurring by non-allelic homologous recombination (NAHR) mediated by LCR22s. In this study, optical mapping has been used to elucidate LCR22 structure and variation in 88 individuals in thirty 22q11.2DS families to uncover potential risk factors for germline rearrangements leading to 22q11.2DS offspring. Families were optically mapped to characterize LCR22 structures, NAHR locations, and genomic signatures associated with the deletion. Bioinformatics analyses revealed clear delineations between LCR22 structures in normal and deletion-containing haplotypes. Despite no explicit whole-haplotype predisposing configurations being identified, all NAHR events contain a segmental duplication encompassing FAM230 gene members suggesting preferred recombination sequences. Analysis of deletion breakpoints indicates that preferred recombinations occur between FAM230 and specific segmental duplication orientations within LCR22A and LCR22D, ultimately leading to NAHR. This work represents the most comprehensive analysis of 22q11.2DS NAHR events demonstrating completely contiguous LCR22 structures surrounding and within deletion breakpoints.